Our research provides valuable insights into the uneven distribution of groundwater copepods in the lowland river valley ecosystem using an integrative taxonomy approach. The study was conducted in 101 wells in the Biebrza River Valley (northeastern Poland), which is one of the largest and best-preserved lowland rivers in Europe. It stands out for its exceptional biodiversity and pristine natural landscapes. Groundwater copepods were found in 49 of the 101 analyzed wells. We identified ten species of Copepoda and eight species of Cladocera. The most frequent copepods were Diacyclops bicuspidatus, Diacyclops crassicaudis, Canthocamptus staphylinus, Paracyclops cf. fimbriatus, Diacyclops bisetosus, and Eucyclops serrulatus. The aforementioned species were categorized as stygophiles, and no stygobionts were detected. Our findings suggest that the groundwater Copepoda community in this region is relatively stable and composed mostly of stygophiles along with stygoxenes. We did not find any significant impact of environmental parameters or different aquifers on the distribution of copepods, suggesting a patchy distribution of groundwater copepods in the lowland river valley. The relatively high presence of stygoxenes suggests that the exchange of organisms between surface water and groundwater plays a vital role in maintaining the diversity of microcrustaceans in lowland river valleys. Our study contributes to filling the knowledge gap regarding groundwater fauna in lowland Europe, particularly in areas affected by Pleistocene glaciations.